On Mobile Genetic Elements in Enterococci; Adding More Facets to the Complexity

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FACULTY OF HEALTH SCIENCES DEPARTMENT OF MEDICAL BIOLOGY

On Mobile Genetic Elements in Enterococci;

Adding More Facets to the Complexity

Eva K. Bjørkeng

A dissertation for the degree of Philosophiae Doctor

July 2010

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On Mobile Genetic Elements in Enterococci;

Adding More Facets to the Complexity

By

Eva K. Bjørkeng

A thesis submitted according to the requirements for the degree of Philosophiae Doctor

Research group for Host-Microbe interactions Department of Medical Biology

University of Tromsø

July 2010

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The presented work was carried out in the period from September 2005 to July 2010 at the Research group for Host-Microbe Interactions, Medical Faculty at the University of Tromsø.

I acknowledge the University of Tromsø for funding my PhD degree. I am also deeply grateful for the extended founding I received from the University hospital to finish my work.

I am most indebted to my supervisors. Without you there would be no thesis.

There have not been many questions that you could not answer or help me find an answer Kristin – I am deeply grateful for your understanding, your knowledge and your patience.

You have the ability to calm your students when times are rough and you always had time for my sometimes rather silly questions. I am thankful for having been one of your students.

Arnfinn – for improving my writings considerably and giving the clinical insights to my subjects – thank you for choosing me for this project.

Johanna – you had always time for my questions and you always share of your great knowledge. Your door is always open for me to ask questions.

I thank Eirik for being almost as a supervisor for me and Erik for saving my BAC project.

I am gratified to Trine and Bettina for their everyday lab-magic.

I thank all the great people in my lab, at the biofilm group and K-res. You all made my days fun, interesting, inspiring, and was always there for good questions and discussions. Thank you - for friendship and your support trough the years.

I am grateful for the three greatest men in my life; Emil, Magne and Glenn for holding out all this time and for your understanding☺. Thank you for letting me know there are other things to life than enterococci and giving me the great life that I have. You are the world to me….

Thanks to my family.

Eva – you have been the bestest of friends and the most supportive one could ever wish for.

You knew exactly how I was feeling and knew how to cheer me up. Thank you for being who you are – and nothing more….☺

I am grateful for all whom I have shared office space with – Torill, Veronika and Malachy

…..you have been good discussion partners

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2000 B.C. – Here, eat this root

1000 A.D. – That root is heathen. Here, say this prayer.

1850 A.D. – That prayer is superstition. Here, drink this potion.

1920 A.D. – That potion is snake oil. Here, swallow this pill.

1945 A.D. – That pill is ineffective. Here, take this penicillin.

1955 A.D. – Oops....bugs mutated. Here, take this tetracycline.

1960-1999 – 39 more "oops"...Here, take this more powerful antibiotic.

2000 A.D. – The bugs have won! Here, eat this root.

— Anonymous

October 2010 Eva Bjørkeng

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Presented papers

Paper I

Eva Bjørkeng, Gunlög Rasmussen, Arnfinn Sundsfjord, Lennart Sjöberg, Kristin Hegstad, and Bo Söderquist. Clustering of polyclonal VanB-type vancomycin resistant Enterococcus faecium in a low-endemic area was associated with CC17-genogroup strains harbouring transferable vanB2-Tn5382 containing pRUM-like plasmids with axe-txe plasmid addiction systems. Submitted

Paper II

Eva Katrin Bjørkeng, Girum Tadesse Tessema, Eirik Wasmuth Lundblad, Patrick Butaye, Rob Willems, Johanna Ericsson Sollid, Arnfinn Sundsfjord, and Kristin Hegstad. ccrABEnt serine recombinase genes are widely distributed in the Enterococcus faecium and Enterococcus casseliflavus species-groups and expressed in E. faecium. Re- submitted Microbiology

Paper III

Bjørkeng E. K., Hjerde E., Lundblad E. W., Sollid J. E., Sundsfjord A., and Hegstad K.

Sequence analyses of an approximately 100-kb chromosomal element supporting transfer of vanB2-Tn5382/Tn1549 from Streptococcus lutetiensis to Enterococcus faecalis and E.

faecium. In manuscript.

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Abbreviations

ccr chromosomal cassette recombinase

GAS Group A Streptococcus

GBS Group B Streptococcus

GCS Group C Streptococcus

GEI Genomic Island

GGS Group G Streptococcus

GI gastrointestinal

GRE Glycopeptide Resistant Enterococci

HGT Horizontal Gene Transfer

ICE Integrative Conjugative Elements

IS Insertion Sequence

MGE Mobile Genetic Element

MIC Minimum Inhibitory Concentration

MLST Multi Locus Sequence Typing

MRSA Methicillin Resistant Staphylococcus aureus

PBP Penicillin binding protein

PCR Polymerase chain reaction

PFGE Pulsed Field Gel Electrophoresis

SCC Staphylococcal Cassette Chromosome

ST Sequence Type

TA Toxin-Antitoxin

VRE Vancomycin Resistant Enterococci

VRSA Vancomycin Resistant Staphylococcus aureus

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1. Introduction

1.1 Enterococcus 1.1.1 Taxonomy:

Enterococci are Gram positive bacteria occurring as single cocci or in chains. They are lactic acid producers and many also produce bacteriocin. Enterococci are non-spore forming, facultative anaerobic organisms, catalase negative and have a remarkable ability to withstand extreme environments which includes high pH, temperature and salt concentrations (75,164).

In addition they are relatively resistant to chemical disinfectants such as chlorine, gluteraldehyde and alcohol. These qualities are important for survival and spread in the hospital environment (28,80,127).

In 1899 Thiercelin first described the name “entérocoque” referring to its intestinal origin and its spherical shape (214). However the enterococci were until 1984 subgrouped into different streptococcus groups. The name was revised when Streptococcus faecalis and Streptococcus faecium was transferred from the genus Streptococcus to Enterococcus due to new molecular information (121,132). The enterococci share phylogenetic relationship with the streptococci.

Both genera are also related to the Lactococcus sp (103). The phylogenetic relationship between enterococci is showed in Figure 1.

According to List of Prokaryotic names with Standing in Nomenclature (LPSN) there are at least 40 species included in the Enterococcus genus (http://www.bacterio.cict.fr/e/enterococcus.html. 2010.25.02). The two most clinical relevant species are the Enterococcus faecium and Enterococcus faecalis which cause the major part of human enterococcal infections (228).

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Figure 1. Phylogenetic tree showing the relationships among atpA sequences from different enterococcal strains. Bootstrap values after 1,000 repetitions are indicated. Reprinted from (172) with permission from

E. faecium-species group:

E. faecium E. durans E. hirae E. ratti E. villorum E. mundtii

E. casseliflavus-species group:

E. casseliflavus E. flavescens E. gallinarum

E. faecalis-species group:

E. faecalis E. moraviensis E. haemopheroxidus

E. cecorum-species group:

E. cecorum E. columbae

E. avium-species group:

E. avium E. maldoratus E. gilvus E. raffinosus E. pseudoavium E. hermanniensis

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1.1.2 Habitat

Enterococci are part of the normal intestinal flora in birds, humans, and animals. They can also colonize the oral cavity and vagina in humans (131). In addition these bacteria are found in the soil, on plants, surface water, and other environments exposed to human or animal faeces (75,136). In the last two decades enterococci have gained increased awareness as important nosocomial pathogens. The main enterococcal infections include urinary tract infections, infections in the intra-abdominal cavity, and endocarditis (24).

1.1.3 Clinical significance

The majority of clinical enterococcal infections are, as mentioned, caused by E. faecalis and E. faecium. E. faecalis is considered more virulent however, E. faecium is more likely to be resistant to antibiotics, even those of last resort (241). Twenty years ago only 10% of the nosocomial enterococcal infections was caused by E. faecium (241). Now around 40% of the enterococcal nosocomial infections worldwide are caused by E. faecium (145,228). This ratio started to change in favour of E. faecium in the US during the late 1990s and in Europe around the year 2000 (130,162,218,219). In the last two decades the emergence of enterococci as an important nosocomial pathogen has been increasingly documented (140,188). The relative proportions of E. faecalis and E. faecium in Norwegian blood culture isolates in 2008 was 4% and 1.4% respectively (NORM 2009.

http://www.unn.no/getfile.php/UNN-Internett /Fagfolk/www.antibiotikaresistens.no/NORM- 09/NORM%20NORM-VET%202008.pdf).

The pathogenesis of enterococcal infections is only partly understood. However, several adhesins, hemolysin, hyaluronidase, aggregation substances, gelatinase, and genes encoding pili are now considered possible virulence factors (75,79,108,110,199). So far, at least 22 different genes, collectively called fms (E. faecium surface protein-encoding genes) are considered putative virulence factors in E. faecium (202). Virulence factors encoded by acmfm

(fms8), hyl, espfm, sgrA and ecbA, are most strongly associated with clinical lineages in E.

faecium (23,45,81,110,171,204).

Putative E. faecium virulence genes and products, their epidemiology and potential role in pathogenesis are listed in Table 1. Resistance in enterococci has increasingly become a problem. This issue will be discussed in section 1.3.

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Table 1. Overview of putative virulence genes in E. faecium, their virulence and epidemiology Virulence gene Pathophysiology/Virulence Epidemiology References

Esp Biofilm formation

Pathogenesis of rat endocarditis Pathogenesis of mouse urinary tract infections

Antigenic in humans during endocarditis and bacteremia

Specifically linked to hospital-associated Enterococcus faecium (Efm) on pathogenicity island

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General Meeting of the American Society of Microbiology, Posters B-167 and B-211 (141)

(139) Acm

(Fms8) MSCRAMM¹

Binding to collagen type I, IV Pathogenesis of rat endocarditis Antigenic in humans during endocarditis

Widespread among

hospital related Efm (171) (170) (169)

Pili PilA

(orf1904/fms21) PilB

(orf2569/ebpCfm)

MSCRAMM

Predicted to be involved in pilus biogenesis.

Widespread among clinical

isolates of Efm (109)

(129)

Scm

(orf418/fms10) MSCRAMM

Binding to collagen type V and to a lesser extent binding to collagen type I and fibrinogen.

Widespread among Efm (203)

EcbA (orf2430) MSCRAMM

Binding to collagen type V Binding to fibrinogen

Specifically linked to HA³

Efm (108)

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Hyl Not known (putative glycoside

hydrolase)

Promotes colonization of mouse gastrointestinal tract

Specifically linked to hospital-associated Efm

(190) (225) (187) (82) Orf903 (fms11) ²CWAP/MSCRAMM High incidence in clinical

strains (108)

(204) Orf2010 (fms14) CWAP/MSCRAMM High incidence in clinical

strains (204)

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efaAfm Cell wall adhesisn High incidence in clinical

strains

(67) SgrA (orf2351) MSCRAMM

Binding to nidogen and fibrinogen. May play a role in biofilm formation

High incidence in clinical

strains (110)

1MSCRAMM – microbial surface component recognizing adhesive matrix molecules

2CWAP - cell wall-anchored proteins

3HA – hospital adapted. Table modified from (199) with permission from the publisher

1.1.4 Population structure and global dissemination

The spread of hospital adapted clonal complexes of E. faecium is considered the major cause for the spread of vancomycin resistant enterococci (VRE) globally (240). These lineages are most often resistant to ampicillin and ciprofloxacin, and contain a large transferable genomic island (140,229,235) and genes for certain virulence markers such as enterococcal surface

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present (82,130). Adhesion to extracellular matrix proteins and formation of biofilms on medical devices such as catheters and stents are important properties of enterococci and add up to their pathogenicity (239). It seems that the enrichment of virulence genes may have added to the success of E. faecium and opened for newer hospital adapted clones (202). The clonal complexes have most likely evolved as a result of multiple recombination events rather than mutations and possibly stress-inducing conditions in the hospitals favouring selection of these clones (13,240). By subsequent acquirement of transposons containing the vancomycin resistance clusters vanA or vanB these VRE have become a basis of pandemic potential (240).

Until recently, all E. faecium clonal complexes were considered to have a common ancestor and they all clustered into a single common clonal complex, CC17 (Figure 2). However it may seem that this single complex in fact are several hospital adapted clonal complexes that have distinct founders (e.g. ST17, ST18, ST22 and ST78 and more) and has developed independently in the same niche. The sequence-types ST17, ST18 and ST78 seem to be the predominant CC17-genotypes worldwide containing vanA and vanB (22,23,45,93,128,130,133,134,140,145,149,181,207,216,225,236,240). E. faecium of animal origin has been considered host specific and not particularly related to human lineages.

However, ampicillin resistant E. faecium belonging to the CC17-genogroup has recently been isolated from pigs and dogs (20,56,57,60) indicating that the hospital lineages are not restricted to the hospital environment anymore. Putative virulence genes were also detected in isolates from dogs (57) The development of these hospital adapted lineages appears to be largely associated with horizontal gene transfer and the ability to readily acquire resistance genes and virulence determinants (85). In addition a higher number of accessory plasmids, such as Inc18 and pRUM, linked to vancomycin resistance, have been detected in CC17- related strains (106,195). A study performed on isolates of E. faecium from US hospital patients at different time periods clearly indicates that the evolution of successful hospital adapted clones have been linked to successive horizontal gene transfer of virulence markers and resistance determinants (85).

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Figure 2. E. faecium Clonal Complex 17 genotype, shown in circle, is the common way of classifying the typical VRE in hospital outbreaks. Reprinted from (57) with permission from the publisher.

1.1.5 The E. faecium genome

The recent developments in pyrosequencing techniques have created a new era for extensive sequencing and characterization of the gene collection of bacterial isolates. The technology allows thorough analysis of diversity and population structure of clinically important bacteria such as E. faecium (228,238). In 2000 the first draft genome sequence of E. faecium DO was published (77) but the whole genome sequence for this strain has not been finished yet and there is still sequences missing (228). Recently genome sequencing of fifteen E. faecium has been performed due to the new technology (179,229). A study of the sequences of seven additional E. faecium from various sources revealed that there is a large difference in the genome size between the strains and a variable number of large plasmids present (229). Data showed that hospital strains were multi resistant whereas the commensal strains were not. The resistance genes appeared to be mostly located on plasmids and up to 30% of the E. faecium genome indicated to be non-core (228). Phylogenetic analysis revealed a relative small evolutionary distance between six of the seven strains. However there were considerable differences in gene content indicating both gene gain and loss as contributors in the evolution

17

18 16

78

CC17 genotype

22

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proteins in the infectious strains and none in the non-infectious. Seven of these proteins were IS elements which may contribute to the genome flexibility in E. faecium. Presence of the known virulence genes esp, hyl, and acm were present in clinical strains however not in all.

The esp gene was verified to be located on a large transferable putative pathogenicity island (PAI) indicating that the gene is acquired by horizontal gene transfer (HGT). Comparative genomic hybridization studies have also showed that the majority of E. faecium strains of clinical origin could be grouped into specific phylogenetic groups (a hospital clade) related to the Clonal Complex 17-genogroup. However a variable content of MGE between isolates shows that MGE may be important factors contributing to different phenotypic characteristics between CC17-related strains. Genes for IS elements and transposases were shown to be more enriched in the clinical isolates (228,229).

1.2 Streptococcus

Streptococci are commensal bacteria on the mucus membranes of the upper respiratory tract, gastrointestinal tract, genitourinary tract and the skin (103). The streptococci tend to be more susceptible to antibiotics than the enterococci. Streptococci are often classified into three main groups in their ability to haemolyse erythrocytes on a blood agar plate; the beta- haemolytic group, the non-beta haemolytic streptococci and the nutritionally variant streptococci, two of them are highlighted in Figure 3 (163). The beta-haemolytic streptococci are divided into several subgroups based on differences in specific carbohydrate antigens - group A (contains Streptococcus pyogenes) (GAS), group B (Streptococcus agalacticae) (GBS), group C (includes Streptococcus dysgalacticae, Streptococcus equi, Streptococcus zooepidemicus, and Streptococcus constellatus) (GCS), and group G (contains Streptococcus equisimilis) (GGS) are the most common ones. In addition there are group E, F, P, U and V (72). The non-beta haemolytic streptococci include Streptococcus pneumoniae, Streptococcus bovis, Streptococcus lutetiensis, and the viridans streptococci. Most of these belong to antigenic group D (72). The phylogenetic relationship between Streptococcus species are shown in Figure 3. This thesis will focus only on the S. lutetiensis (Group D Streptococcus) (163). Normally the streptococci are susceptible for vancomycin. S. lutetiensis has been found both in animals and isolated from clinical isolates (72,183). In 1996 a S. lutetiensis isolate was shown to hold an approximately 100 kb genomic element containing a vancomycin resistance gene cluster highly homologous to the prototype vanB of E. faecalis V583.

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Figure 3. The phylogenetic relationship among 55 Streptococcus species and the classification of different streptococcal strains based on 16S rRNA sequences. One exception in this group is S. dysgalacticae subsp.

dysgalacticae which is not beta-haemolytic but is included for taxonomical reasons. Reprinted from (72)

Beta -haemolytic streptococci

Non-beta haemolytic streptococci

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1.3 Antimicrobial resistance

The impact of antimicrobial resistant bacteria in terms of cost, morbidity and mortality is increasing worldwide (21,46,88). In this regard it is of importance to reveal factors and mechanisms which are involved in the evolution and spread of such bacteria. Enterococci are considered difficult-to-treat pathogens with inherent clinical resistance to all cephalosporins and sulphonamides as well as low-level resistances to aminoglycosides, lincosamides and trimethoprim. In addition E. faecium have the unique capacity of acquiring high-level resistance to aminoglycosides, ampicillin and vancomycin, the most efficient anti- enterococcal drugs (5,164). Being part of the gastrointestinal flora the enterococci are in a unique situation to receive resistance genes from other commensals, but also transfer these to other and more pathogenic bacteria located in the gastrointestinal tract (70,233). This emphasizes the clinical importance of enterococci as a reservoir for antimicrobial resistance determinants.

1.3.1 Development of antimicrobial resistance

An antibiotic is defined as a substance having a biological, semi synthetic or synthetic origin which shows selective activity against bacteria and may thus be used in treatment of bacterial infections. Antimicrobial susceptibility testing of a clinical isolate is usually based on Minimum Inhibitory Concentration (MIC). The MIC value is the lowest concentration of antimicrobial agent which inhibits the growth of the bacteria. Clinical MIC breakpoints are designed to guide therapy and do not necessarily distinguish between bacteria with and without resistance mechanisms. Thus, epidemiological cut off values (the highest MIC value of the wild-type population) are designed to detect low level resistance mechanisms and monitor resistance development. The clinical MIC breakpointsgenerally divide bacteria into three categories of susceptibility: susceptible, intermediate, or resistant (123). A clinical resistant microorganism is unlikely to respond to even maximum doses of a given antibiotic (71). Antibiotic resistance was described early after the introduction of the first “real”

antibiotic – penicillin (147). Later on, this has been the general pattern of observations.

Within few years after introduction of new antibiotics in clinical practice, resistance tend to emerge (157).

The major part of antibiotics used today and the resistance-genes that gives rise of the resistance in several human pathogens have originated from the environment, especially from the soil, and have existed for a long time in nature. For example genes for penicillin resistance

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vancomycin resistance more than 200 million years ago (231,245). Antibiotic resistance in human pathogens is primarily derived from mutation or horizontal gene transfer (4). The major driving force has been the selective pressure of antibiotics use, in medical therapy, veterinary practice, agriculture and animal farming. However some bacteria do contain intrinsic resistance or reduced susceptibility to some antibiotics. Often these genes coding for resistance may have as a role to inhibit growth of bacterial competitors in the soil (157,231).

The species responsible for infections are not producers of antibiotics themselves (157).

1.3.2 Biochemical mechanisms for antimicrobial resistance

The biochemical mechanisms of resistance are divided into four classes where the bacteria can utilize one or more of these in their defence against antimicrobial agents: i) inactivation/

modification of the antimicrobial target which includes degradation and/or chemical modification, ii) reduced access due to altered penetration and or efflux that actively get rid of the antimicrobial agent before it reaches its target site, iii) altered target or protection of target site in which the antibacterial agent can not bind, iv) metabolic bypass by overproduction or alternative pathway.

Antimicrobial resistance in enterococci can be divided into two classes; intrinsic and acquired resistance.

1.3.3 Intrinsic antimicrobial resistance in enterococci

Intrinsic resistance is due to the lack of target sites for the antibiotic in question or insufficient access of the target site within the cell. This resistance is an intrinsic property encoded by the host chromosome and is a species-related trait (4,217).

Enterococcus is generally considered naturally resistant to cephalosporins due to their low affinity for enterococcal penicillin binding proteins (PBPs). The level of susceptibility other β-lactam drugs varies between enterococcal species (217). In addition, enterococci express resistance to sulphonamides due to their ability to use environmental folate (98,164). Low level resistance of aminoglycosides is due to low uptake of the drugs, and resistance to lincosamides is due to putative efflux mechanisms (14,166,217). E. gallinarum and E.

casseliflavus are in addition intrinsically resistant to low levels of vancomycin due to production of D-Ala–D-Ser ending peptidoglycan side chain precursors for which vancomycin has a lower binding affinity compared to the normal D-Ala-D-Ala side chains (131,217).

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1.3.4 Acquired antimicrobial resistance in enterococci

There are two ways for a bacterium to acquire resistance to antibacterial agents, either through chromosomal mutations or by horizontal gene transfer (HGT), where HGT is considered the major mechanism for spread of antimicrobial resistance (59,196).

The intrinsic resistance to several commonly used antibiotics may have given enterococci an advantage for acquiring new resistance phenotypes (162). High levels of resistance to penicillins associated with β-lactamase activity have been reported in E. faecalis, but is considered to be rare (9). Resistance to ampicillin is most often due to modifications in the expression of or mutations in essential PBP proteins. Resistance to ampicillin is often seen in E. faecium but occurs rarely in E. faecalis (131). Resistance to high levels of aminoglycosides (e. g. gentamicin, streptomycin and kanamycin) is due to three mechanisms: reduced uptake (due to increased efflux and/or decreased cell permeability), alterations at the ribosomal binding sites, or production of aminoglycoside modifying enzymes. Streptomycin resistance is acquired mainly via ribosomal resistance by mutations of the 16S or 23S ribosome or enzymatic modification by aminoglycoside adenylyl transferases (ANT) of the antibiotic.

High-level resistance to kanamycin and gentamicin is obtained by aminoclycoside modifying enzymes such as phosphotransferases (APH) and adenyltransferases (AAC). The most common found in enterocooci are the bifunctional enzyme AAC(6´)-Ie-APH(2´´)-Ic which give resistance to most aminoglycosides except streptomycin. The ermB gene is the major contributor to the development of resistance to macrolides, lincosamides, and streptogramins.

The gene encode a methylase modifying an adenosine residue in the bacterial 23S rRNA. A plasmid encoded chloramphenicol acetyltransferase gene, cat, has been shown in 20-42% of enterococci. Resistance to tetracycline is encoded by tetM and tetN genes among others and is seen in up to 80% of enterococci. Resistance is due to production of a protein interacting with ribosomes in such a way that the ribosome is protected and unaffected by the antibiotic. High- level quinolone resistance is gained trough mutations in the subunit for gyrase (GyrA) and in the ParC subunit of topoisomerase IV. Enterococci are now also commonly resistant to quinolones (8,131).

1.3.5 Glycopeptide/vancomycin resistance in Enterococcus – GRE/VRE

The first reports of vancomycin resistant enterococci (VRE) appeared in 1987 in France (142) and the United Kingdom (224). In 1989 North America reported their first incidence. Within few years New York experienced several major outbreaks of VRE in hospitals (83). Within

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two decades VRE became the third- to fourth-most important hospital-acquired pathogen (234).

The epidemiology of VRE infections is somewhat different in Europe and the US. In Europe VRE are often isolated from domestic animals due to the use of avoparcin, an antimicrobial drug used in husbandry giving co-resistance to vancomycin in the enterococcal flora of the farmers. Avoparcin was forbidden in 1997 however vancomycin resistance genes continuing to persist in the environment function as a source for VRE in the community (24,240). In the US avoparcin was not used. Still, transmission of VRE and nosocomial infections has been more frequent due to higher use of vancomycin in the hospitals (24).

The emergence of glycopeptide resistant enterococci has become a major problem because it leaves few options for treatment. The vancomycin resistance genes are transferable to other species, including S. aureus, and selection pressure for the VRE may give rapid expansion of resistant populations. In addition, once the problem with VRE has established it is difficult to limit (162). Despite the global problem it is important to emphasize that vancomycin resistance is not the only challenge we are facing; the enterococci are increasingly acquiring resistance to macrolides, amphenicols, fluoroquinolones, aminoglycosides, and even new antibiotics such as oritavancin have been selected (8,14,95,144).

1.3.6 Glycopeptide resistance mode of action

The peptidoglycan layer of Gram positive bacteria consist of sugar derivatives which are cross linked and a group of amino acids. The peptidoglycan is multilayered where the sugars are connected by crosslinking. In E. faecalis and E. faecium the crosslink consist of two alanine molecules; D-Ala – D-Ala which serve as targets of glycopeptides (47) (Figure 4).

Binding of vancomycin to the D-Ala-D-Ala inhibit two steps in the formation of the new cell wall. It inhibits the addition of new sugar derivatives and the interaction interferes with crosslinking of the peptidoglycan chains to each other. Consequently, the strength of the cell wall is weakened and the bacteria are now likely to lyse.

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Figure 4; Resistance to glycopeptides caused by the replacement of D-Ala peptidoglycan precursor with a D-Lac precursor makes the glycopeptide unable to bind to the cell wall.

Reprinted from (9) with the permission from NEJM.

Glycopeptide resistance is currently divided into nine different classes (vanA, vanB, vanC, vanD, vanE, vanG, vanL, vanM, and vanN) (106,246) (Table 2). The most prevalent acquired gene clusters are the vanA and vanB.

vanA is the most common form of acquired glycopeptide resistance found among the enterococci. This is also the only type detected in S. aureus so far (47). It is characterized by resistance to high levels of both vancomycin and teicoplanin and is often plasmid mediated. It is carried on transposon Tn1546 or closely related elements (200).

vanB type of resistance is associated with inducible variable levels of resistance to vancomycin and susceptibility to teicoplanin. The vanB genotypes can be divided into three subgroups; vanB1-B3 due to sequence diversity (53). The vanB operon can be located chromosomally or on plasmids and is transferable. The vanB cluster consists of seven van genes mostly as part of a larger conjugative element (185).

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Table 2. Vancomycin resistance in clinical relevant bacteria caused by acquired van-type gene clusters. Adapted and modified from (106) with permission from publisher.

Resistance AQUIRED INTRINSIC

level: High Variable Moderate Low Low

Type: VanA VanB VanD VanE VanG VanL VanM VanN VanC

MIC in mg/L:

Vancomycin Teicoplanin

≥ 16

> 8

≥ 4 0,5-1

≥ 64 4-64

6-32 0,5

12-16 0,5

8 8 2-32

0,5-1 Expression Inducible Inducible Constitutive/

Inducible (vanD2)

Inducible/

(Constitutive) Inducible Constitutive/

Inducible

van ligase gene vanA vanB1-B3 vanD1-5 vanE vanG1-2 vanL vanM vanN vanC1-C3

Modified

target D-alanine-

D-lactate D-alanine-

D-serine D-alanine-

D-lactate D-alanine-

D-serine? D-alanine- D-serine Conjugative

transfer

Yes Yes No No Yes No Yes Yes No

Location Plasmid/

chromosome on transposon(s)

Plasmid/

chromosome

±transposon/ICE^a

Chromosome Chromosome Chromosome on possible ICE

Chromosome? Chromosome

Distribution E. faecium E. faecalis E. avium E. casseliflavus E. durans E. gallinarum E. hirae E. mundtii E. raffinosus S. aureus B. circulans O. turbata A. haemolyticum Paenibacillus Rhodococcus

E. faecium E. faecalis E. casseliflavus E. durans E. gallinarum E. hirae S. epidermidis Streptococcus Clostrdium Ruminococcus Eggerthella

E. faecium E. faecalis E. avium E. gallinarum E.raffinosus Non- enterococcal fecal flora

E. faecalis E. faecalis Non- enterococcal fecal flora

E. faecalis E. faecium E. faecium E. gallinarum - vanC1

E. casseliflavus - vanC2/3

a Integrative and conjugative elements

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The vanB genotype has mainly been seen in hospital outbreaks in Scandinavia and Australia.

(16,17,93,104,218,237). The vanB2 subtype seems to be the dominant vanB genotype in most studies. This dominance is presumably related to its integral location in the conjugative transposon Tn5382-like (51,55,61,100,150-152,159,164,223,234).

1.4 Gene transfer mechanisms and their contributions to the development of antimicrobial resistance in enterococci

1.4.1 Mechanisms

Transfer of genes in bacteria is performed in two ways; vertical and horizontal gene transfer.

Vertical gene transfer is genes inherited from parent cells to their offspring. All successful antibiotics targets single gene products encoding essential, functions in the bacteria. A mutation which only slightly changes the protein to which the antibiotic is directed may well make the organism resistant to that drug. These bacteria will persist in an environment containing the actual antibiotic. However in an environment where exposure to lethal levels of antibiotics is not continuous it is not likely that a single mutation leading to the resistance would become an epidemic problem (210). One exception to this notion seems to be the acquisition of stable high-level fluoroquinolone resistance which is associated with mutations in chromosomal genes involved in DNA-replication. Thus, the acquisition of new DNA by horizontal gene transfer have a relative higher impact on the development of antimicrobial resistance compared to mutations.

Horizontal gene transfer (HGT). Horizontal gene transfer (HGT) is the process of which an organism take up genetic material from a different organism which is not an offspring of that particular organism. This way of acquiring new genetic material has had a great impact on the evolution and genome plasticity of prokaryotic organisms, and is the reason for a great extent of the spread of resistance genes (180,193,206).

Resistance genes can be transferred not only within related species but across major taxonomic bacterial divisions. The genetic information may be transferred intercellular in three major ways; transduction, transformation and conjugation. Not all bacteria are able to perform all three processes, but several free living bacteria is able to use at least two of them (210). Integration of a foreign element is often limited by defence mechanisms exerted by the

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recipient (111,156,173) so gene transfer is only successful if the element is able to replicate in the host by itself or through insertion in the host genome, expressed and an advantage for the recipient.

Transduction. Transduction is a process carried out by temperate bacteriophages which insert into the bacterial chromosome as a prophage. This prophage replicates and may at low frequency pack the host DNA or some of the host DNA with its own into the new prophage.

When the cell lyses the bacteriophage may infect a new cell and the new prophage DNA integrates into the new recipient chromosome. Normally a transduction is only successful in transferring alleles of homologous genes among bacteria which are closely related.

Bacteriophages often transfer toxins and virulence factors and do rarely carry antibiotic resistance (84,210). Bacteriophages in enterococci have been shown, although transduction of resistance genes to enterococci trough bacteriophages in vivo has not yet been revealed it is believed that phages may play a role in the spread of antibiotic resistance in enterococci (29,131,247).

Transformation. Transformation is a process in which naked, exogenous DNA is taken up and recombined into the genome of a competent bacterial cell. This process may facilitate evasion of host defences by mediating pilin switch and transfer of resistance genes or might be a process for acquiring nutrient sources for the cell. Transformation may also be a mechanism in the repair of damaged DNA (205). Enterococci are not known to be naturally competent (131), thus this process is not considered an important factor in the evolution of this genus.

Conjugation. Conjugation is carried out by Mobile Genetic Elements (MGE) such as conjugative plasmids and Integrative Conjugative Elements (ICE) that encode a mating apparatus which facilitate transfer of the cellular DNA from a donor cell to a recipient. This process requires close cell-to-cell contact between donor and recipient and a pore through which the DNA can be transferred and then recombined into the recipient genome or circularized as a plasmid (84,210). Conjugative plasmids can also transfer non-conjugative plasmids by mobilization. MGEs may carry virulence factors and they often contain genes encoding antibiotic resistance. Gram positive bacteria and especially enterococci use conjugation as a system for genetic exchange (43).

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Figure 5; Acquisition of antibiotic resistance genes. Resistance genes (Ab^r) can be transferred to a recipient trough three mechanisms; Transformation (uptake of free, naked DNA), transduction (infection of a bacteriophage) or conjugation (plasmids, ICE) (Modified from Kenneth Todar, Lectures in Microbiology, University of Wisconsin-Madison.

(http://textbookofbacteriology.net/themicrobialworld/bactresanti.html )

1.4.2 Mobile genetic elements

Mobile genetic elements (MGEs) are DNA fragments that mediate their own mobilization within or between cells (84). A vast variety of mobile genetic elements has been identified and new elements are continuously being found. The conjugative plasmids and transposable elements will be highlighted below.

Conjugative plasmids

In the bacterial cell the plasmids are replicating autonomously and controlled trough a negative control system; however, they may sometimes integrate into the bacterial chromosome. Several plasmids are self-transmissible or may be mobilized by other plasmids

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making them able to spread to other hosts and provide them additional traits such as virulence and antibiotic resistance.

In enterococci a plasmid classification system based on the replication (rep) genes has been developed. Currently 19 rep families has been identified (119)

Conjugative plasmids encode proteins necessary for their own transfer from a donor to a recipient cell. These proteins involve the pili or the aggregation factors which promote cell-to- cell contact. Before plasmid transfer, a nick is engaged at oriT (origin of transfer) by a relaxase, followed by transfer of a single DNA strand by a rolling-circle or theta-replication, trough a type IV-like secretion system, and synthesis of the second strand in both donor and recipient. Several genes are required for transfer of a plasmid. These includes genes for relaxase, the formation of a mating channel (transglycosylase), genes for energetics of transfer (ATPases), and genes for production of coupling proteins (34,94). Figure 6 shows the organization of the transfer region in the broad host-range plasmid pIP501 as an example.

Figure 6; The pIP501 transfer region. Reprinted from (1) with permission from the publisher

The conjugative transfer of plasmids in enterococci is often divided into two major groups;

pheromone responsive plasmids and broad host range plasmids;

Pheromone responsive plasmids – The communication process of pheromone responsive plasmids seems restricted to E. faecalis (rarely described in E. faecium) and involves the excretion of different small hydrophobic peptide sex pheromones which are specific for different types of plasmids and act as interbacterial signals. The pheromones are produced by

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plasmid is switched on. The result is production of an aggregation substance on the donor-cell surface mediating cell-cell contact between donor and recipient. The donor can now transfer the pheromone responsive plasmid to the recipient. Once the plasmid is transferred expression of this particular sex pheromone will be turned off in the recipient. Other bacterial species do also produce peptides similar to pheromones. For example S. aureus may induce enterococci to enter the aggregative state which may start intergenetic spread of plasmids. However transfer of vancomycin resistance by this mechanism has not yet been shown (44,94,165,178).

Broad host range plasmids – are plasmids that are able to transfer between enterococci and other Gram positive organisms including streptococci and staphylococci. The frequency of transfer is generally much lower than with the pheromone responsive plasmids. Since staphylococci, streptococci, and enterococci share several resistance genes, the broad host- range plasmids may well be a vehicle through which some of these resistance genes have spread among these different genera of bacteria (165).

Most broad host range plasmids are identified in streptococci and enterococci and often hold resistance genes to a wide spectrum of antibiotics. The lower limits in size for these types of plasmids are in the range of 15-20 kb which is the size of the transfer region with no additional genes (94).

Transposable elements

Transposable elements are defined as “specific DNA segments that can repeatedly insert into one or more sites in one or more genomes” (Roberts et al., 2008). It is a diverse group of mobile genetic elements of which some are capable of excising from the bacterial chromosome, transfer to a recipient by conjugation and insert into the recipient chromosome.

The simplest transposable elements are the Insertion Sequences (IS) which are small < 2.5kb DNA elements. They encode a transposase which is an enzyme that cuts the transposable element out and unite it into the DNA where it will be inserted by homologous- or non- homologous recombination between short repeats (48).

Transposable elements may be mobilized by other conjugative elements; in some cases this includes elements in the host chromosome (194). However transposable elements do not hold the phenomena of incompatibility, so conjugative transposons may interact with each other and with other replicative and non-replicative elements. Different variations and

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of a mosaic of larger and more complex elements giving several benefits to the host (194).

After the sequencing era started new and more complex variants of elements are frequently discovered and described. These complex variants make it difficult to classify the transposable elements and several attempts have been proposed during the years. One way of classifying these elements is shown in Table 3. The composite transposons, unit transposons, integrative and conjugative elements and mobile genomic islands will be highlighted below.

Composite transposons – These are DNA segments, often carrying one or more resistance genes to antibiotics, flanked by two separate IS elements of the same IS family which may or may not be exact replicas. Instead of each IS element transposing separately, the whole length of DNA spanning from one IS element to the other is moved in one unit. Composite transposons have been reported in enterococci. The most widespread is the Tn5281 (identical to Tn4001 in Staphylococcus) which hold resistance to high levels of aminoclycosides. The prototype Tn5281 (Figure 7) is flanked by two copies of IS256. In Enterococcus the IS elements within the IS256 and IS1216 families often form composite transposons (106).

Another example of a composite transposon found in enterococci is Tn5385 which is a large (65 kb) complex element containing both Tn5281 and Tn5384, holding resistance to erythromycin, aminoglycosides, mercuric chloride, streptomycin and penicillin (191)

Unit Transposons – Unit transposons encode often an enzyme which is engaged in the excision and integration of the element. These are often site-specific recombinases or resolvases. In addition the elements include one or more accessory genes in their genetic unit and are often flanked by inverted repeats (IR) (193). An example of a unit transposon is Tn1546 encoding the VanA phenotype in Enterococcus (10,142).

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Table 3. Transposable elements in Gram positive bacteria: definitions and examples.

Type of transposable element^*

Definition Examples in Gram

positive bacteria

Reference

Composite transposons

Flanked by IS elements. The transposase of the IS element is responsible for the catalysis of insertion and excision

Tn5281, Tn5384, Tn1547

(116,186,192)

Unit transposons Typical unit elements encode an enzyme involved in excision and integration (DD(35)E or tyrosine) often a site-specific recombinase or resolvase and one or several accessory (e.g. resistance) genes in one genetic unit

Tn3, Tn1546

(10,146)

Conjugative

Transposons (CTns) / Integrative

Conjugative elements (ICEs)

The conjugative transposons (CTns) belong to a group known as integrative conjugative elements (ICEs) which carry genes for excision, conjugative transfer and integration within the new host genome. They carry a wide range of

accessory genes, including antibiotic resistance

Tn916, Tn1545 Tnvamp

(Tn5382/Tn1549),

(36,48,78,86,189)

Mobilisable transposons (MTns)/integrative mobilisable elements (IMEs)

The mobilizable transposons (MTns), also known as integrative mobilizable elements (IMEs), can be mobilized between bacterial cells by other ‘‘helper”

elements that encode proteins involved in the formation of the conjugation pore or mating bridge. The MTns exploit these conjugation pores and generally provide their own DNA processing functions for intercellular transfer and subsequent transposition

Tn4451 (2)

Mobile genomic islands

Some chromosomally integrated genomic islands encode tyrosine or serine site- specific recombinases that catalyze their own excision and integration but do not harbor genes involved in transfer. They carry genes encoding for a range of phenotypes. The name of a genomic island reflects the phenotype it confers, e.g.

pathogenicity islands encode virulence determinants (toxins, adhesins, etc.)

SCCmec (126)

Integrated or An integrated or transposable prophage is a phage genome inserted as part of the φNM1–4 (11,227)

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transposable prophage linear structure of the chromosome of a bacterium which is able to excise and insert from and into the genome

PH15

Integrated satellite prophage

Bacteriophage genome inserted into that of the host which requires gene products from ‘‘helper” phages to complete its replication cycle

IL1403 (39)

Group I intron Small post-transcriptionally splicing (splicing occurs in the pre-mRNA), endonuclease encoding element. Will home to allelic site

Unnamed group I intron inserted the DNA polymerase gene of Bacillus subtilis phage SPO1

(90)

Group II intron Small post-transcriptionally splicing (splicing occurs in the pre-mRNA), restriction endonuclease encoding element

Tn5397 containing group II intron

(161)

Istron Chimeric ribozyme consisting of a group I intron linked to an IS605 like transposase

CdlSt1 (30)

Intein Small post-translational splicing (splicing occurs in the polypeptide), endonuclease encoding element. Will home to allelic site

LLP-KSY1 PolA The intein database

http://www.neb.com/neb/inteins.html

* Not all reported elements have shown to be mobile. Adapted and modified from (193) with permission from the publisher.

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Figure 7. Schematic presentation of the three transposons representing the transposon groups transferring resistance genes in enterococci. Adapted from (106) with permission from publisher.

ICEs (Integrative and conjugative elements)

ICEs are elements which excise by site-specific recombination and integrate like prophages but transfer in a covalently closed circular intermediate by conjugation like plasmids. Before transposing to a recipient these elements needs to excise and form a circular intermediate.

This is a process mediated by site-specific recombinases. At times they may also require an excisionase. The transfer mechanism is thought to be similar to that of plasmids, though with a specific oriT (origin of transfer) (194).

The circular intermediate is replicated during conjugation but replication is not required for maintenance of the element. However the intermediate is not self-replicating and will require replication factors from the host (78). Three types of recombinases have been found to be involved in insertion and integration of these elements; tyrosine, serine and DDE recombinases (32,35). Several of the tyrosine recombinases catalyse site-specific integration into genes encoding tRNA (35). Most of the ICE encodes a tyrosine integrase which catalyse

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would lead to the excision of a circular intermediate containing an attI site which can transfer to a recipient by conjugation. The element will be able to integrate at an attB site (31). The conjugative elements may facilitate transfer of additional DNA by including genes or by integrating into other elements where they may take advantage of the self-transmissibility of the ICE (76,215).

One of the best known ICEs is the Tn916-Tn1545 family. Tn916 from E. faecalis was the first conjugative transposon to be identified (32,43,78). This 18kb transposon is widely distributed among Gram positive bacteria and especially in Enterococcus and Streptococcus. Tn916 was shown to insert into various sites on a plasmid, pAD1 and at several sites in the chromosome of a recipient bacteria (43). Within the Tn916/Tn1545 family there has been identified several different ICE like the Tn5382/Tn1549-like. Tn5382/Tn1549 has shown to be approximately 34kb encoding vanB2 in enterococci. It has been shown that Tn5382/Tn1549 forms a circular intermediate; and transfer of the element has been shown (55,86,138). However it is not established whether this element is conjugative or mobilizable. An oriT and relaxase of the MOBp7 family has been identified. It has been proposed that Tn1549 has been transferred from Clostridium symbiosum to Enterococcus by a helper element (221). A vast diversity of ICE elements has been shown. Several of these are shaped by extensive recombination and formation of mosaic genomes formed by inter-ICE recombination and tandem array structures (87,243).

The streptococcal ICESde3396 shares several open reading frames with a putative ICE from S. agalacticae (GBS), S. dysgalacticae (GGS), and several genes from S. pyogenes (GAS). In addition, ICEde3396 harbours genes from non-streptococcal bacteria such as Enterococcus and Lactococcus, giving the element a mosaic structure in an 18kb internal region. This MGE is able to transfer to other β-haemolytic streptococci and has been shown transferable to other GGS, GBS and GAS.This ICE element does not contain genes for antibiotic resistance rather it includes genes for cadmium and arsenate resistance and a virulence gene encoding agglutinin receptor precursor (58). This example illustrates the dynamics and spread of such elements.

Mobile genomic islands – Genomic islands (GEI) are part of the flexible bacterial gene pool and are typically discrete DNA elements between closely related bacterial strains. The

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size (10-200kb), are often inserted at tRNA genes, may be unstable, and may have a different codon usage and GC content than the core genome. GEIs are often flanked by 16-20bp perfect or almost perfect direct repeats (DR) arisen by site specific integration into the target site and do often act as recognition sites for excision. These elements contribute to a great extent to the diversification and adaptation of microorganisms and have a large impact on the genome plasticity and evolution, dissemination of antibiotic resistance and virulence genes, and formation of catabolic pathways. One hypothesis is that the GEIs embrace a family of elements including recognized mobile elements like ICE, conjugative transposons and some prophages (97,122). Genomic islands with functions that increase the fitness of the bacteria have probably directly or indirectly been positively selected trough evolution (97). One example of a mobile genomic island is the methicillin resistance (mecA) of MRSA isolates is located within the Staphylococcal Cassette Chromosome (SCC) region of the staphylococcal chromosome containing transposon within transposon features. The mecA gene is not originally found in S. aureus and may have been transferred from coagulase negative staphylococci (CoNS) which may be capable to infer resistance to methicillin. MRSA and other resistance phenotypes are often a result of integration of transposons containing resistance genes for example erythromycin (Tn554). The basic SCCmec is composed of the resistance genes, component of its regulatory region and the transposition genes ccrA and ccrB in combination or the single ccrC. The origin of SCCmec is not known however it is believed that the ccr and mec genes accumulated in CoNS from and unknown source followed by deletions in the regulatory part of the mec genes took place whereby the genes were transferred to S. aureus (7,112,167,212). Recently a SCCmec-like element was identified in a strain of Macrococcus caseolyticus (a commensal bacterium in food animals). This element has not previously been observed and has shown a potential mechanism for the generation of new SCCmec-like elements (220). This may give further knowledge of the origin of SCCmec and related elements.

There are eight different types SCCmec (type I-VIII) described so far and each type may vary, showing a diversity of this mobile genomic island (62,249). Even the ccr genes are varying up to 5% at nucleotide level within one type SCCmec (101,176). The whole region of SCCmec can excise from the chromosome through the gene products of the ccr genes making SCCmec a presumable mobile element (189). The detailed mechanism around transfer of SCCmec is not yet fully understood.

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Pathogenicity islands (PAI) are genomic islands present on the genomes of pathogenic strains but are not found in the genomes of non-pathogenic members of the same or related species.

These were first described in human pathogens of E. coli but have been found in the genomes of various pathogens of humans, animals, and plants, both Gram positive and Gram negative (97). An example is the E. faecalis PAI coding for virulence genes including the gene for cytolysin toxin and the enterococcal surface protein (esp). In addition there are traits suspected to contribute to pathogenicity or altering E. faecalis relationship with the host, including a bile acid hydrolase and carbohydrate utilization pathways (158). An interesting part of this PAI is the diversity of the island between closely related strains even between strains belonging to the same clonal complex. McBride and co-workers hypothesize that the pathogenicity island in E. faecalis has entered piece by piece and not as a whole, complete island which later rearranged into the diversity of islands we see today. The genome of E.

faecium has also shown to contain PAIs and other related elements (229). Recently an E.

faecalis PAI was shown to transfer via mechanisms independent of ICE functions. Transfer of this particular PAI only occurred via pheromone responsive type of conjugative plasmids and showed horizontal transfer of all chromosomal regions including a vancomycin resistance transposon and alleles included in MLST (155).

1.4.3 Host range of mobile elements

Mobile DNA disseminates both within a bacterial species but also between different species and genera dependent on the host range of the element. Some elements are even able to transfer from bacteria to eukaryotes (e.g. Agrobacterium tumefaciens).

Plasmids. As mentioned above, plasmids may have a broad host range making it able to spread to several species, but others are restrained to certain bacterial species. It has also been shown that the enterococcal plasmid pAM830 has facilitated transfer of a vanA Tn1546-like element to S. aureus. This plasmid was lost. Broad-host range plasmids in enterococci have shown to transfer between several Gram positive bacteria. Different rep families display different host ranges. An example is rep family 7 and 10 which has shown to transfer to staphylococci, streptococci and Bacillus (119).

Bacteriophages. Bacteriophages, like all viruses exhibit a defined host range. Some are viewed to have a narrow host range and highly specific, whereas others infect a range of

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different bacterial species. Examples of a broad host-range bacteriophage is phage 812 which infect several staphylococcal species (124).

Little is known about the enterococcal phages and their ability to spread virulence traits or antibiotic resistance however they contribute to a large extent to the genome diversity in E.

faecium (120,175,229).

The bacterial viruses are highly adaptive and continuously co-evolving with its hosts (230) and a particular bacteriophage may transfer a DNA element to different bacterial species.

Certain genetic elements may be transduced by several different phages and thus disseminate to several different bacterial species (154).

Integrative and Conjugative Elements. The host-range of a self conjugating element will depend on the specificity of the recombinases/integrases responsible for the conjugation.

Tn916 from E. faecalis has shown to have a very broad host range (more than 50 different bacterial species from 24 genera) including both Gram positive and Gram negative bacteria (19,43).

ICESt1 and ICESt3 in Streptococcus thermophilus has shown to be able to transfer from E.

faecalis. These elements have also been found in S. pyogenes showing the evidence that they may move between different species. The two ICE elements are also closely related to putative ICEs in S. pyogenes, S. mutans, and S. agalacticae. In addition one of the elements is carrying cadmium resistance genes closely related to plasmids from Lactococcus lactis, Listeria innocua, S. aureus, Staphylococcus saprophyticus, and ICEs of Listeria monocytogenes, S. agalacticae, S. dysgalactigae, and SCCmec genomic island from Staphylococcus (18,26,177).

The mixing of genes in ICEs and other genetic elements would seem to be a central mechanism for the exchange and spread of genes into different ecological niches (18).

1.4.4 Integration sites

The integration of a transposable element is not a random process. Enzymatic activity is required for integration of a transposable element. In prokaryotes these enzymes are mostly transposases, integrases and recombinases which recognize specific regions in the target DNA and promote homologous or non-homologous recombination between them (84). Most elements show some target specificity having preferences over some targets than others. The specificity may as well be the structure rather than the sequence of the target DNA, that is

On Mobile Genetic Elements in Enterococci; Adding More Facets to the Complexity

On Mobile Genetic Elements in Enterococci;

Adding More Facets to the Complexity

Eva K. Bjørkeng

On Mobile Genetic Elements in Enterococci;

Adding More Facets to the Complexity

By

Eva K. Bjørkeng

A thesis submitted according to the requirements for the degree of Philosophiae Doctor

Research group for Host-Microbe interactions Department of Medical Biology

University of Tromsø

July 2010

Contents

Presented papers

Abbreviations

1. Introduction

CC17 genotype